Granulocyte metabolism in workers at a coke-processing plant]

Acid phosphatase activity examinations and nitrotetrazolium blue reduction test after latex stimulation in peripheral blood neutrophils were performed in the group of 32 coking plant workers. Reference group consisted of 150 men not exposed to any toxic substances. Neutrophils of coking plant workers, comparing with the reference group, revealed decreased activity of acid phosphatase and impaired reduction of nitrotetrazolium blue after latex stimulation. The results suggest the possibility of toxic influence of coking plant environment on the lysosomal enzymes and oxygen-dependent metabolism of neutrophils what subsequently may diminish bactericidal powers.     

Evaluation of the phagocytic activity of peripheral blood neutrophils in patients with hyperthyroidism

In 80 women with hyperthyroidism (40 with diagnosed Graves disease and 40 with hyperactive nodular goiter) the following tests related to the function of peripheral blood neutrophils have been carried out: 1. nitrotetrazolium blue (NRT) reduction test. 2. evaluation of phagocyting activity by using latex particles and living bacteria cells, and 3. determination of immunoglobulin concentration and the C3 component of the complement in blood serum. The following features were found in the patients with hyperthyroidism: 1. the elevated values of the index of spontaneous NBT reduction which return to normal following the treatment with propranolol or metizol lasting 14 days, 2. a decrease in the phagocyting activity of neutrophils occurring with stimulation of phagocytosis by both the latex particles and bacteria cells. 3. the return to normal values of the index of neutrophils phagocyting the latex particles following two-week treatment with propranolol or metizol. It was concluded that in patients with hyperthyroidism the changes in NRT reduction and phagocyting activity of peripheral blood neutrophils return to normal following the two-week treatment of these patients with both propranolol and metizol.     

Peroxydase activity of the granulocytes in the course of surgical treatment of neoplasm of the alimentary tract

Granulocyte peroxidase activity of patients with alimentary tract neoplasms has been investigated by using the histochemical method. Blood samples were collected before surgical treatment and anaesthesia, during surgical intervention from vessels draining the neoplasms, and 2-3 weeks after surgery from peripheral vessels. The activity of myeloperoxidase in the polymorphonuclear granulocytes of patients was evidently lower in comparison to the activity of this enzyme in the granulocytes of normal individuals. The excision of the tumour did not evidently increase the tested activity in blood from peripheral vein in comparison to the same sample and to the blood from vein draining of tumour. We can suggest that granulocytes may release myeloperoxidase to the surrounding media in the course of neoplasmatic diseases and in this case the cells are exhausted in this enzyme.     

Phagocytic activity of granulocytes after UV-irradiation

The effect of UV-irradiation on three functions of human isolated granulocytes (viability, phagocyte activity and capacity to reduce nitrotetrazolium blue) was investigated. The phagocytosis appeared to be the most sensitive function. The number of phagocytic cells was decreased under UV-doses as low as 0.63 kJ.m-2. Cell lethality was increased under UV-doses 4.32 kJ.m-2 and higher. The capacity to reduce nitrotetrazolium blue was uneffected even at the dose as high as 10.17 kJ.m-2.     

Differentiation of HL-60 promyelocytic leukemia cells monitored by flow cytometric measurement of nitro blue tetrazolium (NBT) reduction

Reduction of nitro blue tetrazolium (NBT) to insoluble blue formazan granules occurs during the stimulus-induced respiratory burst of mature granulocytes and is routinely used as an indicator of the extent of granulocytic differentiation of HL-60 acute promyelocytic leukemia cells. In the present study, the differentiation of HL-60 leukemia cells induced by dimethylsulfoxide (DMSO) or retinoic acid was monitored by flow cytometric (FCM) measurement of forward and 90 degree light scatter of NBT treated cells. Two-parameter correlated analysis permitted a distinction between cells with increased forward and decreased 90 degree light scatter (NBT-), and cells with decreased forward and increased 90 degree light scatter (NBT+). Fixation of NBT treated cells with 1% paraformaldehyde facilitated flow cytometric analysis, and allowed differences in NBT reduction to be quantitated. DMSO-induced cells expressed an all-or-none reduction of NBT to formazan, compared with retinoic acid treated cells that exhibited a graded response. Three parameter flow cytometric analysis of HL-60 leukemia cells stained with propidium iodide in combination with NBT allowed the determination of the cell cycle distribution of NBT-treated cells.     

The depressing effect of tumour on bactericidal capacity of plasma and leukocytes.

Leukocyte--plasma suspensions and plasma from tumour patients have been tested in vitro against staphylococcus aureus for bactericidal capacity. Blood was collected during surgical intervention from peripheral vein and from the nearest point of vein to tumour. Second collection of peripheral blood was done 2--3 weeks later after surgery. Decrease of bactericidal activity of plasma has been found in blood collected from vein near the tumour. We can suggest that tumours release some factors disturbing the bactericidal capacity or that the tumour absorbs some factors of plasma or changes the functions of granulocytes.     

Production of superoxide ions by leukocytes of the American alligator (Alligator mississippiensis)

This study was conducted to characterize the production of superoxide ions by leukocytes in whole blood of the American alligator (Alligator mississippiensis). We used WST-1, a tetrazolium salt which can be reduced to a water-soluble formazan compound with high molar absorptivity at 438 nm, to probe the production of superoxide by alligator leukocytes. Incubation of alligator whole blood with WST-1 resulted in a time- and concentration-dependent increase in absorbance of the plasma at 438 nm. The reduction of WST-1 was inhibited in a concentration-dependent manner by superoxide dismutase, an enzyme that catalyzes the reduction of superoxide to peroxide, confirming that the reduction of WST-1 was due to the presence of superoxide. Treatment of whole blood with nitrotetrazolium blue (NBT) resulted in the staining of heterophils and monocytes, enforcing the idea that that the production of superoxide is due to the presence of leukocytes, and not other blood cell components. It is interesting to note that the production of superoxide by the alligator leukocytes required no external stimulation while human leukocytes must be stimulated with an immunological challenge before producing superoxide. This is the first report of the production of superoxide as an innate immune mechanism in crocodilians.     

Effect of interferon on human neutrophilic granulocytes

The in vitro influence of interferon (IFN) on various functions of human neutrophilic granulocytes was investigated. It was observed that the attachment and engulfment of opsonized yeast particles by human neutrophilic granulocytes were enhanced after preincubation in vitro with IFN for 30 min. The same result was obtained whether the particles were opsonized with fresh normal serum (complement) or with specific antibodies. However, after incubation of the granulocytes with IFN for 3 h the phagocytosis rate was somewhat decreased. Nitroblue tetrazolium (NBT) reduction by resting granulocytes was slightly, although not significantly, increased by preincubation with IFN for 30 min, but their NBT reduction during phagocytosis of E. coli was significantly increased. No major effects of preincubation with IFN were observed on spontaneous or random migration of granulocytes.     

Effect of interferon on human neutrophilic granulocytes

Summary The in vitro influence of interferon (IFN) on various functions of human neutrophilic granulocytes was investigated. It was observed that the attachment and engulfment of opsonized yeast particles by human neutrophilic granulocytes were enhanced after preincubation in vitro with IFN for 30 min. The same result was obtained whether the particles were opsonized with fresh normal serum (complement) or with specific antibodies. However, after incubation of the granulocytes with IFN for 3 h the phagocytosis rate was somewhat decreased. Nitroblue tetrazolium (NBT) reduction by resting granulocytes was slightly, although not significantly, increased by preincubation with IFN for 30 min, but their NBT reduction during phagocytosis of E. coli was significantly increased. No major effects of preincubation with IFN were observed on spontaneous or random migration of granulocytes.     

In vitro studies to prove the effect of sera of eosinophilia in colony formation

Serum samples from four patients with reactive eosinophilia and two patients with eosinophilic leukaemia were compared with normal sera with respect to formation of eosinophil colonies after addition of the sera to mononuclear cells from peripheral blood of healthy subjects. Supernatants from ConA stimulated guinea-pig spleen cells and human lymphocytes were tested in a similar way. Grown colonies were placed on glass slides and after staining with luxol fast blue the percentage of eosinophils was counted. The serum samples of the patients with reactive eosinophilia produced the greatest number of eosinophil colonies while supernatants of spleen and lymphocytes produced the greatest number of eosinophilic granulocytes. Our findings suggest the existence of a factor stimulating eosinophil colonies in the tested serum fractions. Beyond that an indication is given for a substance in the supernatants of spleen and lymphocyte suspensions which stimulates more intensively the maturing into eosinophilic granulocytes than the formation of colonies.     

Influence of levamisole on the capacity of neutrophil granulocytes for ingestion and for the reduction of nitrobluetetrazolium

With the aid of a modification of the NBT test it has been shown in in vitro and in vivo studies that levamisole does not affect the NBT reduction capacity of neutrophil granulocytes. The increased NBT positivity of these cells under the influence of levamisol is due to stimulation of ingestion.     

Indicators of spontaneous and stimulated nitroblue tetrazole test of polymorphous-nuclear leucocytes in acute dysentery patients

140 healthy individuals and 93 sick with acute dysentery were subjected to an examination by spontaneous and by bacterial preparations stimulated reaction with nitroblue tetrazole (NBT test). Indicators in healthy persons were normal in the spontaneous, and increased in the NBT test, stimulated by bacterial preparations. Indicators of the spontaneous NBT test in patients with acute dysentery were raised with a maximum in the period of early convalescence. Stimulation by a live shigella culture--the dysentery vaccine--revealed by means of Sonne diagnostic high, and when endotoxin from Serratia marcescens and dysenterin was used as an inductor, mild indicators of NBT test activity. When a polyvalent agglutinating dysentery serum was used as a stimulator, the activity increased considerably, and a simultaneous use of serum and vaccine had an inhibiting effect on the indicators of the stimulated NBT test. The obtained results testify the sufficient high reserve possibilities of leucocytes towards complete phagocytosis and the efficiency of the NBT test, stimulated by bacterial preparations for the study of functional and metabolic activity of leucocytes in the process of acute bacterial dysentery.     

Cytoenzymatic studies on neutrophils in workers having contact with organic solvents containing benzene, toluene and xylene

In 106 workers (47 women and 59 men) being in professional contact with organic solvents containing benzene and its homologues during 1 to 122 months the cytochemical examination of peripheral blood neutrophils has been performed. The patterns of neutrophil functional activation have been noted expressed in increased activities of acid phosphatase and beta-glucuronidase, increased NBT reduction and diminished glycogen reserves. Those changes were accompanied by diminished peroxidase and alkaline phosphatase activities. The stimulated NBT reduction, elevated in majority of workers, exhibited negative correlation with the exposure time what indicates the practical value of that test monitoring the biological effects of professional contact with the solvents.     

Superoxide-dependent and superoxide-independent pathways for reduction of nitroblue tetrazolium in isolated rat cardiac myocytes.

Spectroscopic studies indicated that nitroblue tetrazolium (NBT) could be reduced to blue formazan by several distinct reactions in suspensions of isolated rat cardiac myocytes. Both NADPH- and NADH-linked pathways for reduction of NBT were observed. NADPH-linked NBT reduction showed little activity in the absence of digitonin, but could be stimulated an average of 9.5-fold by digitonin permeabilization of the plasma membrane. NADH-linked NBT reduction occurred in the absence of digitonin, and could be increased an average of 3.5-fold by digitonin treatment. Analysis of the effects of cell viability on the extent of digitonin stimulation with these substrates suggested that the NADPH-linked reaction involved a cytosolic component, while the NADH-linked reaction involved an intracellular membrane enzyme system. With either NADPH or NADH, NBT reduction was completely inhibited by dicoumarol (100 microM). Dicoumarol-insensitive NBT reduction could subsequently be observed following the addition of 2 mM cyanide, a level of cyanide known to inhibit cytosolic superoxide dismutase. Cyanide-stimulated, dicoumarol-insensitive NBT reduction was augmented by the presence of either antimycin or doxorubicin, two agents which enhance superoxide formation by different mechanisms. The results indicate the existence of multiple pathways for both superoxide-independent and superoxide-dependent reduction of NBT. Dicoumarol-insensitive, cyanide-stimulated NBT reduction may be useful as a spectroscopic probe for intracellular superoxide formation.     

Biological effects of cigarette smoking assessed with the NBT test

The NBT reduction spontaneous and stimulated test was carried out on 90 men; 30 of which had never smoked cigarettes, 28 subjects have smoked for up to 10 years and 32 subjects--since over 10 years. The investigations demonstrated show that in subjects smoking cigarettes for up to 10 years the spontaneous and stimulated ability of the neutrophils to reduce NBT was increased whereas in the subjects smoking cigarettes since over 10 years impairment of the stimulated NBT reduction was demonstrated. We've come to a conclusion that an exposure to tobacco smoke may activate oxidation-reduction processes of the neutrophils in the initial stage and impair them in longer stage of smoking.     

Does ceftazidime or latamoxef affect the phagocytic capabilities of human granulocytes?

In comparative studies made in patients with pneumonia we have treated 14 cases with Latamoxef and 16 cases with Ceftazidime. We have focused attention on the side effects of these drugs and on the phagocytic capacity of granulocytes. In our patients we have noted a decrease of bactericidal activity of plasma and granulocytes and an elevated NBT reduction capability of resting granulocytes. The other tested function of the granulocytes exhibited only small variations. The applied therapy caused the bactericidal activity to be increased in granulocytes and plasma. Both drugs had no harmful effect on the phagocytic activities of granulocytes.     

Neutrophil function screening in patients with chronic granulomatous disease by a flow cytometric method.

Neutrophils from patients with chronic granulomatous disease (CGD) fail to produce a significant oxidative burst following stimulation. We have evaluated the use of flow cytometry and the dye 2',7'-dichlorofluorescein diacetate (DCF) for routine screening for deficiencies of neutrophil oxidative burst. A range for DCF fluorescence for phorbol myristate acetate stimulated and non-stimulated neutrophils was established based on data from 52 healthy adults. Samples from three patients with suspected neutrophil dysfunction, three patients with X-linked CGD, and one patient with autosomal recessive (AR) CGD were evaluated with both the DCF assay and the quantitative nitroblue tetrazolium dye reduction (NBT) test. For the DCF test, the ratio of mean fluorescence intensity of stimulated to non-stimulated neutrophils was less than 5 for CGD patients and from 16 to greater than 50 for healthy individuals. With the DCF test, two populations of neutrophils could be identified in samples from four carriers of X-linked CGD, although two carriers of AR CGD had NBT and DCF results in the normal range. Our data suggest the DCF test is a sensitive and convenient method for detecting CGD.     

Ability of granulocytes to reduce nitroblue tetrazolium in uremic patients treated conservatively

NTB reduction test, both spontaneous and stimulated with E. coli endotoxin, was performed in peripheral blood granulocytes of 40 individuals of both sexes aged between 18 and 64 years treated conservatively at the nephrologic outpatient clinic. Serum creatinine, urea and uric acid were assayed at the same time. A control group included 40 healthy individuals of both sexes aged between 20 and 56 years. Statistically significant increase in spontaneous reduction of NTB was achieved in the group of the uremic patients in comparison with the control group. Moderately positive correlation between creatinine level and percentage of NTB-positive cells in the spontaneous test was shown. Possibility of granulocyte stimulation by uremic toxins is being considered.     

The functional characteristics of the peripheral blood granulocytes in erysipelatous inflammation

The receptors (FcR, C3R) and functional activity, determined by the nitroblue tetrazolium (NRT) test, of polymorphonuclear leukocytes (PML) of low and normal density were studied in erysipelas patients. The leukocytes were obtained by sedimentation on the 2-stage gradient of Ficoll-Verographin (1.077 and 1.119 g/cu cm). No statistically significant difference in the average group indices between "light" and "normal" PNL of erysipelas patients were detected. In comparison with donor PNL, higher expression of C3R, a high spontaneous NBT(+)-PNL level and poor response to stimulation with IgG in the NBT test were observed on granulocytes of the patients. The short-term treatment of the whole blood obtained from the patients with Streptococcus haemolyticus allergen led to a significant increase in the output of "light" PNL. As negative control, brucellin treatment was used, which produced no essential effect. The treatment of donor blood with the above-mentioned antigens did not significantly affect the density of PNL. These facts suggest that in erysipelas the presence of "light" PNL is linked not with the release of granulocytes from the marrow, but with the activation of leukocytes by the products of infective inflammation.     

Phagocytosis and killing of Staphylococcus aureus by granulocytes in mice after granulocyte-macrophage colony stimulating factor (GM-CSF) injection

GM-CSF is a hematopoietic growth factor. In vitro it stimulates the proliferation of myeloid progenitors and formation of granulocyte and macrophage colonies. It was found that GM-CSF in vitro is also stimulated the function of mature granulocytes, but we have no information about such influence in vivo. The purpose of this investigation was the evaluation in vivo of the GM-CSF effect on phagocytosis, bactericidal activity, and lysosome enzyme activities in granulocytes. GM-CSF was injected into mice subcutaneously during 5 consecutive days in the dose of 1 microgram/kg/d. The examination of the percent of cell phagocytizing bacteria (Staphylococcus aureus), NBT test, bactericidal activity and activation of acid phosphatase, alkaline phosphatase, peroxidase and esterase was performed every day and an evident increase of the tested parameters was found. These results prove in vivo activation of granulocytes by GM-CSF.